Glycoproteins are multipurpose glycoconjugates present in all forms of life. The ramifications of their prevalence in biological studies involving cell surface membranes are under active investigation. Glycoproteins are currently believed to be key factors in cellular function and communication. In the present invention, certain highly purified erythrocyte glycoproteins have been identified as the antigenic substances responsible for the hemolysis and hemagglutination of appropriate erythrocytes by suitably adsorbed serum in methods for the detection of infectious mononucleosis (IM) antibody.
Paul and Bunnell reported in their now historic piece of investigative research that non-specific antibodies in the form of hemolysins and agglutinins from sheep cells were present in abnormally high concentrations in the serum of patients during the acute stages of infectious mononucleosis. Am. J. Med. Sci. 183, 90-104 (1932). These sheep cell agglutinating antibodies were heterophilic in nature but were not of the "Forssman-type". Heterophilic antibodies were defined as those having the ability to react with a number of antigens that were apparently unrelated to those that stimulated their production. The "Forssman-type" antibodies were defined as those that were reactive with the erythrocytes of sheep and the heterophilic antigens produced by guinea pig kidney [Davidsohn, I. and Walker, P. H., Am. J. Clin. Path. 5:445-465 (1935)]. Many assays that detect heterophile antibodies have been developed. See, e.g., U.S. Pat. Nos. 3,426,123 and 3,708,572.
Stuart et al and Beer reported that horse erythrocytes also reacted with the heterophile antibodies found in sera of patients with infectious mononucleosis. See Proc. Soc. Exp. Biol. 34: 212, 215 (1936) and J. Clin. Invest. 15:591-599 (1936).
These observations have provided the basis for the immunochemical diagnosis of infectious mononucleosis through the isolation and purification of glycoprotein from mammalian erythrocytes.
Workers have reported an infectious mononucleosis receptor isolated from sheep erythrocyte stroma. The receptor is a glycoprotein whose antigenic determinant is closely related to the presence of sialic acid. [Callahan, H. J., Int. Archs. Allergy Appl Immun. 51:696-708 (1976)]. This investigator used a hemoglobin containing stroma which was denatured in the reaction mixture such that hemoglobin contamination could never be completely removed from the glycoprotein product. The initial sheep erythrocyte preparation was prepared by a hot phenol extraction method. In the present invention the applicant extracted the glycoprotein from sheep and horse erythrocytes with hot ethanol. The resulting glycoprotein product is pure and has a high reactivity with the IM heterophile antibody.
Glycoprotein fractions have been isolated from horse by chloroform methanol extraction. These fractions were closely associated with certain blood group antigens. [Guertler, K. G., Schmid, D. O., Yebou, D. A. and Cleve, H., Blood Grps. Biochem. Genets. 9:41-45 (1978)]. However, the product obtained was not tested for reactivity with IM or other P-B positive antibodies. A close examination of the reported data revealed that the isolated glycoprotein was probably impure and was hence unsuitable for use in assays as sensitive as radioimmunoassays. Other investigators have reported glycoproteins isolated from various mammalian species and have obtained "purified" glycoprotein. For example, Hamaguchi and Cleve reported a method of isolating glycoprotein from human, ox, swine, horse and sheep erythrocytes by chloroform-methanol extraction. However, the isolated extract was crude by comparison to the purified glycoprotein of this invention The serological activity of the Hamaguchi preparation were about 1000-fold less than that of this invention. [See also Fujita, S., Cleve, H., Biochim. Biophys. Acta 406:206-213 (1975); Grant, D. C. Martin, W. G. and Anastassiadis, P. A., J. Biol. Chem. 242(17): 3912-3918 (1967); Hamazaki, H., Hotta, K, Konishi, J., Comp. Biochem. Physiol. 55 B:37-44 (1976)].
An important criterion for the diagnosis of infectious mononucleosis has been based on erythrocyte agglutination or complement mediated hemolysis of blood cells from several species such as goat, horse, bovine and ox.
Bovine erythrocytes were found to have a high specificity for the IM heterophile antibody. Investigators believed that bovine erythrocytes contained immunological determinant(s) which bound more specifically to heterophile antibodies than the immuno-determinants present in erythrocytes from other species [Evans, A. S., et al J. Infect. Dis. 132:546-554 (1975)].
As early as 1971, the present inventor and coworkers [Fletcher, M. A., and Woolfolk, B. J., J. Immunol. 107:842-853 (1971)] found that the partial purification of bovine erythrocytes with 75% ethanol yielded a crude glycoprotein extract which showed high reactivity with heterophile antibodies. The reactivity was based on the result of each of three test methods which measured hemagglutination inhibition, quantitative precipitation and agar gel diffusion.
Several diagnostic tests for IM have been reported that are based on bovine erythrocyte antigens and which employ crude extracts as the purest form of the reactive principle in bovine erythrocytes. For example, see U.S. Pat. Nos. 3,826,821; 3,840,655; 3,959,456 and 4,228,148.
The applicant has purified the bovine erythrocyte membrane glycoprotein to an essentially homogeneous form. The homogeneity of this bovine glycoprotein was shown in several ways, including polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified form of this bovine glycoprotein for IM detection resulted in at least a ten-fold increase in sensitivity in hemagglutination inhibition tests relative to the results obtained with the crude extract of bovine erythrocytes described by Fletcher et al in the year 1971. The results obtained with this glycoprotein in partially purified form were reported on Mar. 27, 1980 by the present inventor and co-workers in Levey B. A. et al, J Clin. Microbio. 11, 256-262 (1980).
The partially purified and homogeneous bovine glycoprotein products have also been described in a pending application: U.S. Ser. No. 247,934, filed Mar. 26, 1981.
The present invention involves the isolation of antigens from horse and sheep erythrocyte glycoprotein which react with IM but not with Forssman antibodies. The IM activity is associated with certain sialoglycoproteins and is completely abolished by neuraminidase treatment. The glycoproteins carry the antigenic determinant responsible for the reaction with the Paul-Bunnell antibody. This characteristic has made possible the development of certain serological diagnostic tests for IM which have heretofore required intact erythrocytes and complicated, time consuming adsorption procedures. See, for example, Levey et al., supra.
An additional receptor property of the glycoproteins isolated here is their ability to interact with peripheral blood lymphocytes forming "E rosettes" in vitro. An "E rosette" is a peripheral blood lymphocyte to which three or more sheep red blood cells have become attached. Rosetting is an accepted marker for human T lymphocytes and thymocytes. In this invention the receptor which is responsible for rosetting has been isolated from sheep and horse erythrocyte glycoprotein. Thus, the purified reagent may be useful in the preparation of a standardizable reagent for the enumeration of rosetting lymphocytes.